Fungal diseases of crop plants have been one of the principal causes of crop losses throughout the history of crop cultivation. The growing of crops as monocultures encourages the proliferation of virulent races of fungal pathogens and wherever a new variety of crop plant becomes grown on a wide scale of the risks of a virulent strain of a pathogen evolving to attack that crop increase drastically. The occurrence of disease is significantly worsened by the international transport of pathogen-carrying plant materials, which can bring together plants with pathogens against which they have had no opportunity to evolve resistance. Thus by man's intervention the natural balance between host and pathogen has been disturbed with disastrous effect on a number of occasions. Catastrophic losses and even famines such as occurred in Ireland during the 19th century, caused by the potato blight fungus (Phytophthora infestans) have resulted from such activities. Fungal disease can also make it completely impossible to grow certain crops in large areas, as was the case when Fusarium wilt wiped out tomato growing in large areas of the Eastern USA or the downy mildew (Plasmopara viticola) fungus devastated vine growing in parts of Europe. Outbreaks of fungal disease can also have a severe effect on the environment as happened when almost the entire English Elm (Ulmus procera) population was destroyed by Dutch Elm Disease (Ceratocystis ulmi). In addition the losses which may be caused during the growing of crops fungal disease may contribute to further post harvest losses. Various soft rots such as Botrytis cinerea are particularly problematic in soft fruit, for example. The fungus Aspergillus flavus, although not a true disease-causing fungus, causes post-harvest rot on stored peanuts and maize, especially in tropical countries and is most serious because it produces a toxin, aflatoxin, which is very toxic to man.
The major economic problems associated with fungal diseases are found in wetter parts of the World, principally Western Europe and the humid tropics. Various crop husbandry techniques, such as crop rotation and avoiding the spread of soil on machinery etc., are used to prevent the build up and spread of severe infestations of fungal disease. Plant breeding has made a significant impact on improving the resistance of many crops to important diseases. For example, plant breeders successfully introduced resistance genes 1 and 1-2 effective against Fusarium oxysporum f.sp. lycopersici into tomato. Nonetheless, problems remain, particularly when many forms of race-specific resistance break down as new races of the pathogen rapidly evolve. In tomato, another virulent strain of F. oxysporum has occurred and breeders are seeking a third useful resistance gene. In cereals growing in parts of Western Europe a recent outbreak of a virulent strain of yellow rust (Puccinia striiformis) has lead to a rapid increase in fungicide use on varieties which remain resistant to other fungi. In these specific cases chemicals are widely used to control fungal disease, as in cases where there are simply no natural sources of resistance available to the breeder.
Chemical fungicides remain a major input in the costs of crop production in many parts of the World. In 1990 21% of all agrochemical sales were accounted for by fungicides (US $ 5.54 million). Farmers and growers have a strong motivation to reduce their input costs. Added to the economic justification is an increasingly strong environmental component in the equation. There is growing pressure in the more advanced economies, notably in North America and Western Europe, from politicians and consumers for agriculture which relies less on chemical inputs. The justification for such demands may lack focused rationale or scientific proof, but fears grow with reports of pesticides traced in groundwater or detected in quantities exceeding the minimums acceptable as food residues. In The Netherlands, for example, there is a mandatory requirement to reduce total pesticides use by 50% before 2000.
Phytophthora infestans belongs to the group of fungi referred to as Oomycetes. Phytophthora infestans infects various members of Solanaceae, such as potato, tomato and some ornamentals. It causes late blight of potatoes and tomatoes affecting all parts except roots. Geographically, the fungus is widely distributed, and it can be found in all potato-producing countries. Economically late blight in potatoes is of major importance, as infection early in the season can severely reduce crop yield. Currently the disease is controlled by spraying chemical fungicides (dithiocarbamates, such as mancozeb, manec and zineb) regularly. Both from an environmental and economical point of view, biological control of diseases caused by Phytophthora infestans could have advantages over the use of chemical fungicides.
Pythium also belongs to the group of fungi referred to as Oomycetes. The genus Pythium differs from the related genus Phytophthora by forming relatively undifferentiated sporangia. Geographically, this fungus is widely distributed on all continents. The first main type of disease caused by Pythium species is damping-off, due to sudden and fast developing attacks on young seedlings in the field or in nurseries. Pythium species cause a second type of disease which is root necrosis and causes a general slowing of plant growth (for example wheat and maize) and loss of yield. The main losses caused by Pythium in Europe are to field crops such as sugarbeet. In principle, losses tend to be all-or-nothing. Similarly, nursery sowings of ornamentals and forest trees may be completely destroyed. (For a review on Oomycetes, vide: European Handbook of Plant Diseases, ed. by I. M. Smith et al., 1988, Blackwell Scientific Publications, Ch.8)
Another fungus is Botrytis, especially B. cinerea, belonging to the group of Fungi Imperfecti, which causes gray mold blight or bud and flower blight, which is common on soft ripe fruits after harvesting, but it can also occur before harvest. It can also affect various vegetables such as lettuce, beans and tomato. Other species of Botrytis are common on flowers, such as lilies, gladiolus and tulips.
A protein with antifungal activity, isolated from TMV-induced tobacco leaves, which is capable of causing lysis of germinating spores and hyphal tips of Phytophthora infestans and which causes the hyphae to grow at a reduced rate, was disclosed in WO91/18984 A1. This protein has an apparent molecular weight of about 24 kDa and was named AP24. Comparison of its complete amino acid sequence, as deduced from the nucleic acid sequence of the AP24 gene, with proteins known from databases revealed that the protein was an osmotin-like protein.
Despite initial success in combating fungal pathogens, such as Phytophthora infestans, and the genetic engineering of plants capable of producing these antifungal proteins with activity against this fungal pathogen there remains a need to identify and isolate other proteins with antifungal activity against this fungus.